ABSTRACT Our R01 project supports NIAID?s mission to better understand, treat, and prevent infectious diseases by focusing on pre-erythrocytic malaria vaccine development. Vaccines that efficiently stop the Plasmodium sporozoite (spz) or liver stage provide complete protection against malarial disease and will enable eradication efforts. There are currently no FDA-approved malaria vaccines for use in humans although repeated dosing with intravenously-administered attenuated spz has shown sterile protection against challenge in multiple Phase 1-2 clinical trials. Recently, CD8+ T cells that reside in the liver (liver resident memory T cells or Trm cells) have been identified as key cell types in protection against liver stage infection. Vaccine strategies that increase liver Trm cells and can be readily adapted to clinical use are therefore critically needed. Such vaccines could bolster CD8+ T cell immunity and may result in T cell-focused vaccines that achieve durable, high-grade protection for persons in endemic and non-endemic regions. Our laboratory has developed a two-dose vaccine that increases liver Trm cells and achieves sterile protection. This approach requires only a single dose of spz. This project aims to provide pre-clinical support for development of this two-dose ?prime-and-trap? vaccine. The University of Washington (UW) will collaborate with established partners at Sanaria Inc. In Aim 1, we will evaluate biological and technical questions about the proposed vaccine regimen, including dose dependence, effects of spz cryopreservation, adjuvant effects, interference from pre-existing antibodies, and use of multiple DNA plasmids. In Aim 2, we will investigate the magnitude and degree of antigen spreading following vaccination, a phenomenon that could enhance protection in the liver. In Aim 3, we will evaluate the prime-and-trap vaccine in the P. knowlesi non-human primate (NHP) immunization-challenge model and demonstrate Trm cell targeting in a P. falciparum NHP model. Tolerability and toxicology endpoints will be obtained in NHP studies in preparation for future clinical studies. In summary, this project will optimize and assess a two-dose prime-and-trap vaccine rationally designed to elicit complete protection against the Plasmodium liver stage.